The use of fluorescent reporters to study cells and tissue is known and a body of work has grown up around this technique. Most of this work, however, deals with ex vivo samples and application to living tissue in vivo is difficult given the fluorescence microscopy tools presently available.
The use of therapeutic antibodies (“Ab”) in cancer treatment is a relatively new endeavor. Several compounds are now approved for use in the United States (e.g., HERCEPTIN, RITUXAN, ZEVALIN) and more are in development. These compounds are restricted to treating refractory diseases and are not presently used as a first-line treatment. A factor in this disposition of Ab therapies is the lack of clear knowledge of effect. To be effective, the Ab therapy should be selective in attaching to the target and stay attached to the target sufficiently long to exert or induce a clinical effect. There is no currently viable or acceptable method by which such measurements of clinical effect can be made in vivo in target tissue.
The phenomenon of fluorescence is well studied and understood. As applied in biology, the focus is generally on choosing compounds that fluoresce at convenient wavelengths, have certain molecular weights, bind to a substrate in a certain way, resist photobleaching and the like For example, many commercial fluors (e.g., the ALEXA series from Molecular Probes, Eugene, Oreg.) are in the 500-900 Dalton range, whereas green fluorescent protein is nearly 30,000 Daltons. The fluorophores can either be conjugated with a substrate molecule, or activated or bound indirectly. Some compounds of interest, e.g., DOXORUBICIN, are naturally fluorescent, though the strength of the fluorescence may not be optimal for monitoring purposes. The ability to probe different fluors at different wavelengths is desired in that it may allow for intricate, multi-faceted labeling studies.
A significant volume of work has been done to map optical properties of tissues in the body with endoscopic techniques as discussed in, for example, Potential New Endoscopic Techniques for the Earlier Diagnosis of Pre-Malignancy by Rollins et al., Best Pract Res Clin Gastroenterol, 15(2):227-47 (2001). In general, researchers have assessed tissue absorption and endogenous fluorescence spectra in an attempt to create characteristic signatures of, say, benign or malignant tissue. For example, adenomatous colon polyps were examined by diffuse reflectance spectroscopy as discussed in Diffuse Reflectance Spectroscopy of Human Adenomatous Colon Polyps in Vivo by G. Zonios et al., Applied Optics 38(31):6628-37 (1999). Autofluorescence spectroscopy has been used for the characterization of esophageal cancer (Light-Induced Autofluorescence Spectroscopy for the Endoscopic Detection of Esophageal Cancer, B. Mayinger, Gastrointest Endosc 54(2):195-201 (2001)), colonic polyps (Colonic Polyp Differentiation Using Time-Resolved Autofluorescence Spectroscopy, M. Mycek, Gastrointest Endosc 48(4):390-4 (1998)), and head and neck cancer (In Vivo Native Cellular Fluorescence and Histological Characteristics of Head and Neck Cancer, S. Schantz, Clin Can Res 4(5):1177-82 (1998)). The introduction of exogenous fluors has also been tried (e.g., Fluorescence Endoscopy of Gastrointestinal Disease Basic Principles, Techniques, and Clinical Experience, H. Stepp, Endoscopy 30(4):379-86 (1998)), especially as related to photodynamic therapy (e.g., Mayinger, 2001). Unfortunately, following detailed kinetics with endoscopic techniques of this sort would likely be commercially undesirable.
Several approaches have been proposed for in vivo optical imaging in small animals. Anti Cancer, Inc. (San Diego, Calif.) has developed an extensive catalog of probes conjugated to green fluorescent protein (GFP) and has produced images of tumors in vivo in nude mice as discussed in, for example, Visualizing Gene Expression by Whole-Body Fluorescence Imaging, M. Yang, Proc Natl Acad Sci 97(22):12278-12282 (2001). However, due to the strong attenuation and scattering of visible light by tissue, this technique is limited to tumors within a few millimeters of the skin surface. Xenogen Inc. (Alameda, Calif.) proposes the use of a bioluminescent reporter (LUCIFERAN) and a sensitive external camera to image subsurface events, such as the effects of antineoplastic drugs on tumor cells as discussed in Visualizing the Kinetics of Tumor-Call Clearance in Living Animals by T. Sweeney, Proc Natl Acad Sci 96(21):12044-9 (1999). Again, this technique is hampered by the attenuation of the light signal by tissue and is thus limited to mice with tumors near the surface. The long integration times required compromise following detailed kinetics. Neither one of these techniques is thought to be transferable to human clinical applications.
Despite the above, there remains a need for systems that can monitor the fluorescence of analytes at clinically useful depths in humans over time.